The effect of gamma irradiation on tetraploid wheat was investigated by studying the effect on growth parameters such as shoot and root growth and the efficiency of energy conversion into growth and the effect on chromatin in the form of chromatin bridges (break in one chromosome arm), ring chromosomes (breaks in both chromosome arms), micronuclei and incomplete mitosis.
Shoot growth rates
Shoot growth rate was investigated to investigate metabolic activity as affected by the gamma irradiation that can be observed over time. Shoot growth of the irradiated seed displayed growth retardation when compared to the control group. Retardation also showed an increase with increasing irradiation dosages (Fig. 1a).
Shoot growth by means of the ANOVA tests revealed highly significant effects of dosage, as well as dosage by time interactions (Table 2).
Shoot growth according to the LSMEANS showed highly significant differences for all pairs of shoot growth, except for the treatment pairs of control and 50 Gy, control and 150 Gy, 50 Gy and 150 Gy, which were non-significant (Table 3).
Root growth rates
Root growth rate was investigated to investigate metabolic activity as affected by the gamma irradiation that can be observed over time. Root growth of the irradiated seed also displayed growth retardation when compared to the control group. Retardation also showed an increase with increasing irradiation dosages (Fig. 1b).
Root growth by means of the ANOVA tests revealed highly significant effects of time and dosage, as well as dosage by time interactions (Table 2).
Root growth according to the LSMEANS showed highly significant differences for all pairs of root growth (Table 3).
Efficiency of energy conversion into growth
The efficiency of energy conversion over time was investigated to observe the effect of gamma irradiation on energy expenditure for growth and repairing of damage caused by the gamma irradiation that change over time. The efficiency of energy conversion into growth declined with increasing irradiation dosage. Hundred and fifty Gy displayed a more efficient conversion into growth than the control and 50 Gy. Fifty Gy and 150 Gy were entangled with the control, while 250 Gy and 350 Gy were at lower levels than the control (Fig. 1c).
Efficiency of energy conversion into growth by means of the ANOVA tests showed highly significant effects of dosage and dosage by time interactions. The time effect was slightly significant (Table 2).
Efficiency of energy conversion into growth according to the LSMEANS revealed that most treatment pairs demonstrated highly significantly differences in efficiency of energy conversion into growth except for the treatment pairs, the control with 50 and 150 Gy that were non-significant, the control and 250 Gy which was significant and 50 Gy with 250 Gy which was non-significant. (Table 3).
Presence of bridges
The presence of bridges in anaphase (Fig. 2a) and telophase (Fig. 2b) was assessed over the period from 17.5 to 47.5 hours after the onset of imbibition to visualise the effect of the gamma irradiation on the chromosomes during mitosis (Fig. 3). In the investigation it was not always possible to determine if a bridge was single stranded or double stranded, therefore, all bridges were assessed together without this distinction. However, evidence of the presence of chromatin breaks due to gamma irradiation first became evident in the form of bridges at 22.5 hours. The number of cells containing different numbers of bridges increased with an increase in irradiation dosage. Fifty Gy and 150 Gy treated cells displayed cells with one to three bridges. The number of cells with more than one bridge became rarer with an increase in number of bridges. Cells with two and three bridges occurred rarely in 50 Gy treated material, while 150 Gy treated material had marginally more of these cells with a substantial increase in cells with a single bridge. 250 Gy and 350 Gy treated material displayed many more cells with chromatin bridges, ranging from cells with one, to cells with seven bridges. Broken bridges were visible in telophase (Fig. 2b) and newly divided cells. Fragments were visible in metaphase (Fig. 2c) and anaphase.
The average number of anaphase and telophase cells containing bridges clearly showed an increase with an increase in gamma irradiation dosage (Fig. 4a). Throughout the assessment period the number of cells displayed periods of an increase and periods with a decrease in the number of cells containing bridges at all dosages.
The extent of DNA damage through gamma irradiation was also assessed by determining the average number of bridges over the investigating period (Fig. 4b). There were large similarities between the average number of anaphase and telophase cells containing bridges and average number of bridges. The similarities decreased with an increase in gamma irradiation. The average number of bridges by means of the ANOVA tests showed highly significant effects of dosage, time and dosage by time interaction (Table 4).
The effect of gamma irradiation on the average number of bridges according to the LSMEANS revealed that the differences among all irradiation treatments were highly significantly different (Table 5).
Presence of ring chromosomes
Ring chromosomes result from chromosomal breaks in both chromosome arms, followed by the fusion of the sticky ends to form a ring chromosome. Ring chromosomes were observable from late prophase (Fig. 2d), through metaphase (Fig. 2e) and in anaphase (Fig.2f). A maximum of two ring chromosomes were observed per cell. The ring chromosomes divide at a later stage in anaphase than the rest of the chromosomes (Fig. 2f).
An investigation of the presence of ring chromosomes in dividing cells revealed that, in general, ring chromosomes appeared earlier in prophase with an increase of gamma irradiation dosage, however, the exception was 50 Gy material, that did reveal ring chromosomes earlier than 150 Gy material (Fig. 5). First appearance of ring chromosomes in metaphase and anaphase followed the same pattern as for prophase, except for 50 Gy material in which ring chromosomes in metaphase and anaphase were observed later than first appearance in prophase. The mean number of ring chromosomes showed a steady increase with an increase in dosage. Most of the ring chromosomes were observed in metaphase cells.
A comparison of the average number of ring chromosomes revealed the average number of ring chromosomes increased with an increase in gamma irradiation dosage (Fig. 6). Fifty Gy material displayed the least number of cells with ring chromosomes and 350 Gy material the greatest number, while 150 and 250 Gy material displayed an intermediate number of cells with ring chromosomes. The presence of ring chromosomes displayed a cyclic pattern with little or no cells with ring chromosomes to peaks of cells with ring chromosomes. The highest peak in 50 Gy material had an average of 0.4 ring chromosomes and the highest peak in 350 Gy material had an average of 3.4 ring chromosomes. It appeared that the number of peaks decreased with an increase of irradiation dosage. Fifty and 150 Gy each had three peaks, while 250 and 350 Gy each had two peaks.
The extent of DNA damage through gamma irradiation was also assessed by determining the total number of ring chromosomes over the investigating period. The total number of ring chromosomes by means of the ANOVA tests showed highly significant effects of dosage, time and dosage by time interaction (Table 5).
The effect of gamma irradiation on the total number of ring chromosomes according to the LSMEANS revealed moderate to highly significant differences among the irradiation dosage treatments except between the 150 Gy and 250 Gy treatments that was non-significant (Table 6).
Presence of micronuclei
Acentric fragments become micronuclei towards the end of telophase when nuclear membranes are formed. Once fragments are formed into micronuclei, they are passed to daughter cells (Fig 2g, h) by chance. They become visible at telophase and are present until prophase. During late prophase, metaphase and anaphase they lose their membranes and are visible as acentric fragments. In this investigation, the greatest number of micronuclei recorded per cell at interphase was seven.
The range and numbers of cells containing different numbers of micronuclei increased with an increase in gamma irradiation dosage (Fig. 7). Fifty Gy material displayed only cells with one or two micronuclei; 150 Gy material displayed cells with one to four micronuclei, while in 250 Gy and 350 Gy material the number of micronuclei ranged from one to seven. In all the treatments, cells with one micronucleus was most frequently, followed by cells with two micronuclei. Cells with three and four micronuclei occurred at lower frequencies, while cells with five to seven micronuclei occurred rarely, less than five cells out of 500.
The total number of micronuclei at various times after the onset of imbibition was determined to obtain an understanding of the effect of gamma irradiation at different dosages (Fig. 8a). The data revealed that there was an increase in the number of micronuclei with an increase in dosage, with a mean number of about 24 micronuclei in 50 Gy material to 310 micronuclei in 350 Gy material.
The total number of micronuclei by means of the ANOVA tests showed highly significant effects of dosage, time and dosage by time interaction (Table 5).
The effect of gamma irradiation on the total number of micronuclei according to the LSMEANS revealed highly significant differences among the dosage treatments (Table 6).
The effect of gamma irradiation on the number of micronuclei was also supported by an increase in the total number of cells containing micronuclei (Fig. 8b) with an increase in gamma irradiation dosage. When the pattern of the total number of micronuclei and the total number of cells containing micronuclei over time were compared, it was found that the developmental patterns were very similar.
Incomplete mitosis
In Triticum turgidum ssp. durum L. the maximum number of nucleoli in the nucleus is four, formed by NOR’s on chromosomes 1B and 6B [20, 21]. Incomplete mitosis was scored as cells with five (Fig. 2i) and more nucleoli in the nucleus resulting from separation of the chromatids during mitosis but still get packed in the same nucleus during interphase, leading to a nucleus in interphase with twice as much DNA [22]. All the irradiation dosages displayed interphase cells with incomplete mitosis from 25 h after the onset of imbibition with an increase of incomplete mitosis with an increase in irradiation dosage. Fifty Gy and 150 Gy had four and three collection times with no incomplete mitosis interphase cells respectively, while 250 Gy and 350 Gy had incomplete mitosis interphase cells at all the collection times from 25h onwards (Fig. 9).
The fact that the first observations of interphase cells with incomplete mitosis was at 25 h, is supported by the fact that many mitotic divisions had completed by that time. It is expected that the changes over time would be different and the effects of the different gamma irradiation dosages would be different. The total number of interphase cells with incomplete mitosis by means of the ANOVA tests showed highly significant effects of dosage, time and dosage by time interaction (Table 5).
The lower irradiation dosages are expected to have little impact on the number of interphase cells with incomplete mitosis, while the higher irradiation dosages will have a larger effect. The effect of gamma irradiation on the total number of interphase cells with incomplete mitosis according to the LSMEANS revealed highly significant differences among the irradiation dosage treatments, except for a non-significant difference between 50 Gy and 150 Gy (Table 6).
Correlations between seedling and root growth and the efficiency of energy conversion on the one side and presence of bridges, micronuclei and interphase cells with incomplete mitosis on the other
Correlations between seedling and root growth and the efficiency of energy conversion into growth on the one side and presence of bridges, micronuclei and interphase cells with incomplete mitosis on the other were determined to evaluate if seedling and root growth are measuring different effects of gamma irradiation than the efficiency of energy conversion into growth does (Table 6).
The relatedness between seedling and root growth with the presence of bridges (Table 6) displayed highly significant correlations between seedling and root growth with the presence of one to three bridges per cell with the highest correlation with two bridges per cell. The efficiency of energy conversion on the other hand displayed highly significant correlations with two to three bridges per cell with the highest correlation with three bridges per cell.
The relatedness between seedling and root growth with the presence of micronuclei (Table 6) displayed highly significant correlations with one to six micronuclei per cell. Seedling growth had the highest correlation with two micronuclei and root growth with one micronuclei. The efficiency of energy conversion into growth on the other hand had highly significant correlations with one to seven micronuclei, with the best correlation with four micronuclei per cell.
The relatedness between seedling and root growth with the presence of incomplete mitosis (Table 6) displayed highly significant correlations with interphase cells with incomplete mitosis. Root growth had the highest correlation with interphase cells with incomplete mitosis, followed closely by seedling growth. The efficiency of energy conversion into growth also had a highly significant correlation with interphase cells with incomplete mitosis, although it was much less than the other two.
Determination of the optimum dosage for mutation breeding
Due to the differences in the correlations between root and seedling growth on the one hand and efficiency of energy conversion on the other with bridges, micronuclei and interphase cells with incomplete mitosis, it was concluded that the efficiency of energy conversion are measuring other aspects of growth retardation.
In Triticum monococcum [8], the 100 Gy and higher gamma irradiation treatments were not entangled with the control like 250 Gy (after 84 h) and 350 Gy gamma irradiation treatments in the present study. The 100 Gy treatment differed from the control with p=0.01 in Triticum monococcum [8]. In the present study 250 Gy differed from the control with p=0.02. This makes it ideal for using p=0.01 for the determination of the ideal dosage for mutation breeding.
Steps taken for the determination of the ideal dosage for mutation breeding. Firstly, the LSD value at p=0.01 must be determined. The LSD value was determined as 7.72. Secondly, the LSD value must be subtracted from 100 to obtain the y-axis value of the graph. The y-axis value was determined as: 100 – 7.72 = 92.28. Thirdly, a best-fit equation must be obtained. A quadratic equation, y=0.0004x2 + 0.0785x + 97.99, had the best fit with R2=0.9218. Fourthly, the x and y values must be plotted according to the quadratic equation with the efficiency of energy conversion into growth on the y-axis and the gamma irradiation dosage on the x-axis. Fifly, by using the 92.28 on the y-axis, the ideal dosage for mutation breeding will be obtained on the x-axis (Fig. 10). The ideal gamma irradiation dosage for mutation breeding was determined as 267.6 Gy.